Abstract Metastatic breast cancer (MBC) is an incurable disease, affecting 10-15% of breast cancer patients, and is often refractory to therapy. Since tumor tissue is not always available after therapy is complete, there is a need for biomarkers that can be monitored noninvasively such as from blood to measure risk of recurrence due to microscopic metastatic residual disease. Numerous studies suggest that DNA methylation could be a useful biomarker for improving the clinical management of disease. Recently circulating cell-free (cf)DNA has attracted attention for clinical use in the context of risk prediction, prognostication and prediction of response to chemotherapy in human cancer and several groups have now reported the detection of tumor-associated DNA methylation patterns in plasma or serum. We identified by whole genome bisulfite sequencing hypermethylation of 21 CpG islands (CGI) in plasma of MBC compared with disease free survivors or healthy cases. Our hypothesis is that a 21-gene DNA hypermethylation signature involving rationally selected hotspots detectable in circulation can be used to detect micrometastatic disease at the end of therapy in patients with early stage breast cancer. In Aim 1 we plan to determine the frequency and the sensitivity and specificity of this DNA methylation signature as a prognostic test in retrospectively collected plasma samples with targeted bisulfite amplicon sequencing. In Aim 2 we will determine the analytical limit of detection and track how different degrees of tumor burden impacts the methylation status of the signature in cell-free DNA in preclinical models of breast cancer metastasis. To clinically validate the biomarker, in Aim 3 we will analyze samples collected as part of prospective study already initiated where we plan to enroll 100 newly diagnosed high-risk breast cancer patients. Blood will be collected after neoadjuvant therapy and surgery and CpG4C in cfDNA will be tested by bAmplicon-seq. We have already collected blood from 20/39 consented patients after their neoadjuvant therapy and subsequent surgery. We will classify patients as positive or negative for CpG4C at the end of neo-adjuvant therapy and additionally at the post-operative blood draw based on cut-off criteria defined in Aim 1 and statistically determine the utility of the marker to prognosticate recurrence. In time such a blood test would also be advantageous at the time of surgery and/or after the completion of chemotherapy to predict which patients could benefit from additional and curative therapies.